New methods for fragmenting biopolymer molecules, mainly peptides and proteins, have recently been developed for use in ion cyclotron resonance or Fourier transforms mass spectrometry (ICR-MS or FTMS). They consist in allowing multiply charged ions to react with electrons, resulting in the fragmentation of the chain-shaped molecules. If one begins with positive ions that are charged by attachment of a few protons, then the neutralization energy of the first proton released in the process leads to the fragmentation of the chain molecules at the precise location where the proton was localized. The method is known as “electron capture dissociation” (ECD for short). If the molecules were originally doubly charged, one of the two fragments created remains as an ion. The fragmentation of proteins and peptides, in particular, follows very simple rules in this process (for specialists: there are predominantly c cleavages, which lead to relatively high ion signals, and only a small number of a and z cleavages between the amino acids of a peptide), so that it is relatively simple to draw conclusions about the amino acid sequence from the fragment ion spectrum. It is significantly easier to interpret these ECD fragment spectra than it is to interpret fragment spectra produced by collision induced dissociation (CID).
For a fragmentation by electron capture, the kinetic energy of the electrons must be low, as otherwise no capture can take place. In practice one supplies electrons with an energy of only a few electron-volts (eV). This procedure is very easy in the extremely strong magnetic fields of the Fourier transform mass spectrometer because the electrons originate from a flat thermion cathode and simply drift along the magnetic field lines with only very low acceleration until they reach the cloud of ions. A second type of electron capture is possible with electrons having a kinetic energy of some 10 to 30 electron-volts (eV). This is termed “hot electron capture dissociation”, or “hot ECD” for short. It results in very similar fragmentation.
It is also possible to fragment triply or multiply charged positive ions in this way, but the method is particularly impressive when used with doubly charged ions. If electrospray ionization is applied to peptides, the doubly charged ions are generally also the most commonly occurring. Electrospray ionization is a method of ionization which is used particularly frequently for biomolecules for the purpose of mass spectrometric analysis in Fourier transform mass spectrometers (FT-MS).
Recent findings have shown that a fragmentation similar to ECD occurs when multiply charged positive ions of biopolymers react with negatively charged ions of low electron affinity, for example with negative ions of Fluoranthene, transferring an electron. Negative radical cations are particularly favorable. Fragmentation by electron transfer is very similar to fragmentation by electron capture. “Electron transfer dissociation” is abbreviated to ETD.
In the case of multiply charged positive ions, reactions between multiply charged positive ions and negatively charged ions can also be used for extensive charge stripping. This is achieved by using negative ions with high electron affinity which do not generate any fragment ions. It is therefore possible to use “charge stripping” to transfer multiply charged protein ion mixtures with broad charge distribution into a mixture which consists almost entirely of singly charged ions. This mixture of singly charged ions can be very easily analyzed in simple mass spectrometers without having to have a complicated charge deconvolution of the mass spectrum obtained.
Ion guides designed as multipole rod systems are usually operated with a two-phase RF voltage, the two phases being applied in turn across the pole rods. The RF voltage across the rods of the rod system is usually not very high. In the case of commercial ion guide systems it is only a few hundred volts at a frequency of several megahertz. In the interior, a multipole field is generated which oscillates with the RF voltage and drives ions above a threshold mass to the central axis, causing them to execute so-called secular oscillations in this field. The restoring forces in the ion trap are sometimes described using a so-called pseudopotential, which is determined via a temporal averaging of the forces of the real potential. In the central axis is a saddle point of the oscillating real potential; this decreases, according to the phase of the RF voltage, from the saddle point to the rod electrodes of the one phase and increases towards the other rod electrodes. The saddle point itself is usually at a DC voltage potential.
These ion guides can be used to transport the ions, and also especially as collision cells to fragment the ions, or as cooling cells for damping the oscillations of the ions. They are normally filled with collision or deceleration gas; after losing part of their kinetic energy the ions then collect in the axis of the system under the influence of the pseudopotential.
Ion guides have also been developed which have a weak DC voltage drop along the axis, thus driving the ions to one end of the ion guide; they especially include ion guides which are not designed as multipole rod systems. Ion guides can also consist of wire pairs coiled in the form of a double helix or quadruple helix, the wire pairs being charged with RF voltages. DC voltage drops across the wire pairs lead to a DC voltage drop in the axis of the system and hence to the ions being driven forward. Ion guides can also consist of a large number of coaxially arranged apertured diaphragms connected alternately to the two phases of an RF voltage. Here, also, it is possible to generate a DC voltage drop along the axis, as already described in the patent prepublication DE 195 23 859 A1 and the equivalent U.S. Pat. No. 5,572,035.
A popular way of ionizing large biomolecules is to use electrospray ionization (ESI), which ionizes the biomolecules out of solutions at atmospheric pressure outside the mass spectrometer. These ions are then introduced into the vacuum of the mass spectrometer by means of inlet systems of a known type. This ionization produces practically no fragment ions, but essentially only ions of the unfragmented molecules, which arise by the attachment of one or more protons to the molecule. The attached protons mean that the mass of these ions no longer corresponds to the mass of the molecules, and so they are frequently termed “quasi-molecule ions”. During electrospray ionization, multiple protonation frequently leads to multiply charged ions of the molecules, depending on the size of the ions. In the case of peptides in the range 800 to 3000 Dalton, doubly protonated ions predominate; with larger molecules, ions with three or more protons prevail. The lack of almost any fragmentation during the ionization process limits the information from the mass spectrum to the molecular weight; there is no information concerning internal molecular structures which can be used for further identification of the substances present. This information can only be obtained by acquiring fragment ion spectra (daughter ion spectra).
Various types of mass analyzers are suitable for analyzing the ions, particularly for analyzing the ion reaction products from positive and negative ions. Time-of-flight mass analyzers with orthogonal ion injection have proven to be outstanding, however.
Time-of-flight mass spectrometers with orthogonal ion injection (OTOF for short) are characterized by a high precision of their mass determination and by a high duty cycle using most of the ions supplied. They operate with a continuous ion beam and normally acquire between 10,000 and 20,000 spectra per second, which can be added to form sum spectra in real time. If one adds only 1,000 spectra over a twentieth of a second, then the mass spectrometer with 20 sum spectra per second can also follow the most rapid chromatographic or electrophoretic separation processes, as are to be expected for chip-based separators. Adding over a longer period increases the dynamic range of measurement. This type of mass spectrometer can be manufactured at moderate cost and is extraordinarily flexible in its application, something which no other mass spectrometer has so far managed to achieve.
If these time-of-flight mass spectrometers are set up as tandem mass spectrometers to scan daughter ion spectra, they have, until now, carried out the fragmentation of selected parent ions to daughter ions using collisions in gas-filled collision cells. The parent ions are usually selected using upstream quadrupole mass filters; RF ion guides are used as collision cells for the fragmentation. The fragment ions obtained as a result of collisions are then injected into the time-of-flight mass analyzer and measured as a daughter ion spectrum.